Bst71I: an isoschizomer of the type-IIS restriction enzyme, BbvI, recognizing the GCAGC(8/12) site.

A new type-IIS restriction enzyme, Bst71I, with the specificity 5'-GCAGC(N)8/3'-CGTCG(N)12 was isolated from Bacillus stearothermophilus (Promega No. 71). This enzyme is an isoschizomer of BbvI with somewhat improved characteristics for use by molecular biologists.     

Isolation and properties of a thermostable restriction endonuclease (ENDO R-Bst1503).

A restriction endonuclease was isolated from Bacillus stearothermophilus1503-4R (Bst1503) and purified to homogeneity. The enzyme required Mg2+ ion as a cofactor. Bst1503 exhibited maximal activity between pH 7.5 and 8.0, between 60 and 65 degrees C, and with about 0.2 mM Mg2+. Bst1503 was not inactivated after exposure at 55 or 65 degrees C for up to 10 h. After 2 h of incubation at 70 degrees C, Bst1503 was inactivated by 65%. Bst1503 was rapidly inactivated at 75 degrees C. A single protein-staining band having a molecular weight of 46,000 was observed when Bst1503 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to exist in two active forms, the predominating form with an S value of 8.3 (180,000) and the second form with an S value of 5.4 (96,000). No conversion between the 8.3S and 5.4S forms was observed after storage. Bst1503 recognized six sites in TP-1C deoxyribonucleic acid (DNA), one site in pSC101 and simian virus 40 DNAs, and three sites in lambdavir DNA. Bst1503 and BamHI were determined to be isoschizomers. The effect of temperatures on the activity and stability of BamHI was determined.     

The methylation status of DNA derived from potato plants recovered from slow growth

The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.     

Cloning, primary structure determination and comparative analysis of DNA-methyltransferases from SfaNI and Bst19I restriction-modification systems

Genes encoding DNA-methyltransferases which recognize the same sequence 5'-GCATC-3' from SfaNI and Bst19I restriction-modification systems have been cloned and primary structures of these have been determined. It has been revealed that restriction-modification system Bst19I contains two DNA-methyltransferases M1.Bst19I and M2.Bst19I, whereas RM system SfaNI include only one DNA-methyltransferase M.SfaNI, N- and C-domain of which are homologous of M2.Bst19I and M1.Bst19I, respectively. M1.Bst19I and M2.Bst19I as well as both domains of M.SfaNI contain conservative elements in an order that is typical for N6-adenine DNA-methyltransferases alpha class. SfaNI and Bst19I DNA-methyltransferases share high homology level with methylases of FokI and BstF5I RM systems. Probably this reflects presence of the common DNA sequence 5'-GATG-3' in the recognition sites of all these RM systems. Basing on primary structures homology of methylases, highly conserved amino acid residues on known spatial model of DNA-methyltransferase M.DpnIIA have been determined.     

Restriction fragment length polymorphism at the HLA-C locus

We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde 1, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLLs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu lI site in the fifth intron.     

Plasmids of the cyanobacterium Synechocystis 6701

Abstract Plasmids were obtained from Synechocystis 6701 using a lysis method that employed a hemicellulase digestion procedure. Eight major bands were observed in the initial preparation. Four of the smaller plasmids were isolated using preparative agarose electrophoresis gels and identified by restriction endonuclease analysis. Chromosomal DNA was screened with 15 restriction enzymes and 6 ( Eco RI, Sst I, Hpa I, Bst EII, Acc I, and Bgl II) were effective. Analysis of DNA fragments from plasmids pSCY 1–4 indicated that each plasmid was unique and that their approximate sizes were 5, 7.5, 13.5 and 15 kb, respectively. Digestion of pSCY 1 and pSCY 4 with Bgl II produced DNA fragments that may be used to construct a conjugation vector for this unicellular cyanobacterium.     

Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute

Ribosomal protein mutations, termed Minutes, have been instrumental in studying the coordination of cell and tissue growth in Drosophila. Although abundant in flies, equivalent defects in mammals are relatively unknown. Belly spot and tail (Bst) is a semidominant mouse mutation that disrupts pigmentation, somitogenesis and retinal cell fate determination. Here, we identify Bst as a deletion within the Rpl24 riboprotein gene. Bst significantly impairs Rpl24 splicing and ribosome biogenesis. Bst/+ cells have decreased rates of protein synthesis and proliferation, and are outcompeted by wild-type cells in C57BLKS<-->ROSA26 chimeras. Bacterial artificial chromosome (BAC) and cDNA transgenes correct the mutant phenotypes. Our findings establish Bst as a mouse Minute and provide the first detailed characterization of a mammalian ribosomal protein mutation.     

Disrupted retinal development in the embryonic belly spot and tail mutant mouse

The Belly spot and tail (Bst) semidominant mutation, mapped to mouse Chromosome 16, leads to developmental defects of the eye, skeleton, and coat pigmentation. In the eye, the mutant phenotype is characterized by the presence of retinal colobomas, a paucity of retinal ganglion cells, and axon misrouting. The severity of defects in the Bst/+ retina is variable among individuals and is often asymmetric. In order to determine the role of the Bst locus during retinal morphogenesis, we searched for the earliest observable defects in the developing eye. We examined the retinas of Bst/+ and +/+ littermates from embryonic day 9.5 (E9.5) through E13.5 and measured retinal size, cell density, cell death, mitotic index, and cell birth index. We have found that development of the Bst/+ retina is notably dilatory by as early as E10.5. The affected retinas are smaller than their wildtype counterparts, and optic fissure fusion is delayed. In the mutant, there is a marked lag in the exit of retinal cells from the mitotic cycle, even though there are no observable differences in the rate of cellular proliferation or cell death between the two groups. We hypothesize that Bst regulates retinal cell differentiation and that variability of structural defects in the mutant, such as those affecting optic fissure fusion, is a reflection of the extent of developmental delay brought about by the Bst mutation.     

Recombinant DNA-methyltransferase M1.Bst19I from <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> 19: Purification,properties, and amino acid sequence analysis

The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5′-GCATC-3′) in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW that carries a tandem of thermo inducible promoters P _R /P _L from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from Escherichia coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature optimum at 50°C and demonstrates maximal activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 ± 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 ± 0.31 μM. Catalytical constant (k _cat) is 1.8 ± 0.05 min⁻¹. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence.     

Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola)

For identification of single species within the Isotoma viridis group, we present polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as a fast and efficient DNA-based molecular method. We used five PCR primers amplifying the cytochrome oxidase II (COII) region (760 bp) of the mitochondrial DNA. The sequences clearly separated four species ( I. viridis , I. riparia , I. anglicana and I. caerulea ) out of samples from Norway, Sweden, Germany and Switzerland. Examination of genetic variation and phylogenetic relationship did not support the separation of two colour pattern forms of I. viridis into distinct species. For RFLP, several restriction enzymes were tested for their ability to produce not only species-specific restriction fragment patterns but to discriminate more than one species per enzyme used with as few cleavage sites as possible. Such a design should render a clear fragment pattern when performing a double digest. These demands appear to be fulfilled best by the combination of the restriction enzymes Mfe I, Nci I and one of Aci I, Bst EII, Nde I, or Sfc I. From the enzymes tested in a previous study, Ase I proved to be reliable, whereas Mbo I can no longer be recommended.     

Restriction enzyme banding of mouse metaphase chromosomes

Fixed metaphase chromosomes from mouse strain RIII embryos or A9 cells were treated with a restriction endonuclease, followed by Giemsa staining. Aha I, Hinf I, or Mbo I treatment produced a C-band pattern, and Eco RII or Hae III produced a G-band plus C-band pattern. Ava II and Bst NI each produced a G-band pattern, but on most chromosomes only a small segment of each C-band, adjacent to the centromere, was stained. These tiny residual C-bands may contain a minor satellite located adjacent to the major satellite clusters.     

The Relationship of PTH Bst BI Polymorphism, Calciotropic Hormone Levels, and Dental Fluorosis of Children in China

The aim of this study was to explore the association of parathyroid hormone (PTH) gene Bst BI polymorphism, calciotropic hormone levels, and dental fluorosis of children. A case-control study was conducted in two counties (Kaifeng and Tongxu) in Henan Province, China in 2005-2006. Two hundred and twenty-five children were recruited and divided into three groups including dental fluorosis group (DFG), non-dental fluorosis group (NDFG) from high fluoride areas, and control group (CG). Urine fluoride content was determined using fluoride ion selective electrode; PTH Bst BI were genotyped using PCR-RFLP; osteocalcin (OC) and calcitonin (CT) levels in serum were detected using radioimmunoassay. Genotype distributions were BB 85.3% (58/68), Bb 14.7% (10/68) for DFG; BB 77.6% (52/67), Bb 22.4% (15/67) for NDFG; and BB 73.3% (66/90), Bb 27.7% (24/90) for CG. No significant difference of Bst BI genotypes was observed among three groups (P > 0.05). Serum OC and urine fluoride of children were both significantly higher in DFG and NDFG than in CG (P < 0.05, respectively), while a similar situation was not observed between DFG and NDFG in high fluoride areas (P > 0.05). Serum OC level of children with BB genotype was significantly higher compared to those with Bb genotype in high fluoride areas (P < 0.05). However, no significant difference of serum CT or calcium (Ca) was observed. In conclusion, there is no correlation between dental fluorosis and PTH Bst BI polymorphism. Serum OC might be a more sensitive biomarker for detecting early stages of dental fluorosis, and further studies are needed.     

Microarray-based evaluation of whole-community genome DNA amplification methods

Three whole-community genome amplification methods, Bst, REPLI-g, and Templiphi, were evaluated using a microarray-based approach. The amplification biases of all methods were <3-fold. For pure-culture DNA, REPLI-g and Templiphi showed less bias than Bst. For community DNA, REPLI-g showed the least bias and highest number of genes, while Bst had the highest success rate and was suitable for low-quality DNA.     

Gene structure and chromosomal localization of mouse <Emphasis Type="Italic">Opa1</Emphasis> : its exclusion from the Bst locus

Background  

Autosomal dominant optic atrophy type 1 (DOA) is the most common form of hereditary optic atrophy in human. We have previously identified the OPA1 gene and shown that it was mutated in patients with DOA. OPA1 is a novel member of the dynamin GTPase family that play a role in the distribution of the mitochondrial network. The Bst (belly spot and tail) mutant mice show atrophy of the optic nerves and previous mapping data raise the possibility that Bst and OPA1 are orthologs. In order to analyse the Bst mouse as a model for DOA, we therefore characterized mouse Opa1 and evaluated it as a candidate for the Bst mutant mouse.     

Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease

A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63 degrees C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application.     

Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing.

Multiplexed amplification of specific DNA sequences, by PCR or by strand-displacement amplification, is an intrinsically biased process. The relative abundance of amplified DNA can be altered significantly from the original representation and, in extreme cases, allele dropout can occur. In this paper, we present a method of linear amplification of DNA that relies on the cooperative, sequence-dependent functioning of the DNA mismatch-repair enzyme endonuclease V (EndoV) from Thermotoga maritima (Tma) and Bacillus stearothermophilus (Bst) DNA polymerase. Tma EndoV can nick one strand of unmodified duplex DNA, allowing extension by Bst polymerase. By controlling the bases surrounding a mismatch and the mismatch itself, the efficiency of nicking by EndoV and extension by Bst polymerase can be controlled. The method currently allows 100-fold multiplexed amplification of target molecules to be performed isothermally, with an average change of <1.3-fold in their original representation. Because only a single primer is necessary, primer artefacts and nonspecific amplification products are minimized.     

Linkage and physical mapping of prolactin to porcine chromosome 7

Comparative mapping studies between human and pig have shown that there is conserved synteny between human chromosome 6 and pig chromosomes 1 and 7, but some gene locations are not well established. Prolactin ( PRL ), an anterior pituitary hormone, has been mapped to human chromosome 6, and has tentatively mapped to pig chromosome 7 using Southern-RFLP analysis with a limited number of meioses. To confirm the assignment of prolactin to porcine chromosome 7 by physical and linkage analysis, pig cDNA and human genomic DNA sequences were used to design pig-specific PCR primers. The primers amplified a fragment of ≈2·8 kb. Two polymorphic restriction sites were identified within this fragment with the restriction endonuclease Bst UI. Prolactin was significantly linked to six markers on the published PiGMaP map of pig chromosome 7. Prolactin was physically mapped using a pig × rodent somatic cell hybrid panel. An analysis of these data placed PRL on pig 7p1·1–p1·2 with 100% concordance and was in complete agreement with the linkage data. Both mapping techniques placed PRL in a conserved order with the loci in the syntenic region of human chromosome 6.     

Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability

D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD(Bp)) at a 2.7-A resolution. The structure of HYD(Bp) consists of a core (alpha/beta)(8) triose phosphate isomerase barrel fold and a beta-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD(Bp) shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD(Tsp)) and Bacillus stearothermophilus (HYD(Bst)), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD(Tsp) contains more aromatic residues in the interior of the structure than HYD(Bp) and HYD(Bst). Changes of large aromatic residues in HYD(Tsp) to smaller residues in HYD(Bp) or HYD(Bst) decrease the hydrophobicity and create cavities inside the structure. HYD(Tsp) has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD(Bp) and HYD(Bst). Besides, HYD(Tsp) also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.