Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.     

Structural basis for thermostability of endo-1,5-alpha-L-arabinanase from Bacillus thermodenitrificans TS-3

The crystal structure of a thermostable endo-1,5-alpha-L-arabinanase, ABN-TS, from Bacillus thermodenitrificans TS-3 was determined at 1.9 A to an R-factor of 18.3% and an R-free-factor of 22.5%. The enzyme molecule has a five-bladed beta-propeller fold. The substrate-binding cleft formed across one face of the propeller is open on both sides to allow random binding of several sugar units in the polymeric substrate arabinan. The beta-propeller fold is stabilized through a ring closure. ABN-TS exhibits a new closure-mode involving residues in the N-terminal region: Phe7 to Gly21 exhibit hydrogen bonds and hydrophobic interactions with the first and last blades, and Phe4 links the second and third blades through a hydrogen bond and an aromatic stacking interaction, respectively. The role of the N-terminal region in the thermostability was confirmed with a mutant lacking 16 amino acid residues from the N-terminus of ABN-TS.     

Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans

A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.     

Molecular cloning of the gene encoding thermostable endo-1,5-alpha-L-arabinase of Bacillus thermodenitrificans TS-3 and its expression in Bacillus subtilis

The gene that encodes a thermostable endo-arabinase (called ABN-TS) from Bacillus thermodenitrificans TS-3 was cloned, sequenced, and expressed in the mesophilic B. subtilis. The gene contained an open reading frame consists of 939 bp, which encodes 313 amino acids. The deduced amino acid sequence of the enzyme showed 50, 46, and 36% similarity with endo-arabinase from B. subtilis IFO 3134 (PPase-C), Pseudomonas fluorescens (ArbA), and Aspergillus niger (ABNA), respectively. The hydrophobic and acidic amino acids making up ABN-TS outnumbered those in PPase-C. The gene product expressed in B. subtilis, as the host, had substantially the same characteristics, and was stable up to 70 degrees C, and the reaction was optimal around 70 degrees C, as well as native ABN-TS.     

Visualization of gene expression in whole mouse retina by in situ hybridization

The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is useful to visualize the expression of specific genes in the entire vascular plexus and retina. However, in situ hybridization on whole-mount retinal preparations is problematic because isolated retinas have a tendency to curl up during hybridization and are difficult to stain. Here we provide a detailed protocol that overcomes these difficulties and visualizes the mRNA distribution of one or two genes in the context of the counterstained retinal vasculature. The protocol takes 3-4 d for single-probe stains, with an additional 2 d for immunohistochemistry co-labeling. In situ hybridization with two probes adds a further 3 d.     

Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme

Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the K(m)s for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5'-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful.     

Antifungal activity of Bacillus sp. isolated from compost

Four strains ofBacillus isolated from lupine compost exhibited an antifungal activity against six plant fungal pathogens (Rhizoctonia solani, Bipolaris sorokiniana, Sclerotinia sclerotiorum, Trichothecium roseum, Fusarium solani, Fusarium oxysporum). It was significantly influenced by the composition of the cultivation media.     

A cryptic plasmid, pAO1, from a compost bacterium, Bacillus sp

The complete nucleotide sequence of a new cryptic plasmid, pAO1 isolated from a compost bacterium Bacillus sp., has been analyzed. Analysis of the PCR-based 16S rRNA sequence showed the bacterium harboring pAO1 was closely related to Bacillus pallidus. The plasmid pAO1 was 3,325 bp in size. Two open reading frames, ORF1 and ORF2, encoding putative polypeptides of 248 and 290 amino acids, respectively, were identified within the sequence. The ORF1 has a limited sequence similarity to an integrase/recombinase, while the ORF2 has high similarity with the replication protein of pBC1 from Bacillus coagulans. A putative origin sequence for a plus-strand was located between ORFs. Southern blot analysis indicates this plasmid replicates via a rolling circle-type mechanism.     

Constitutive heterochromatin polymorphisms in human chromosomes identified by whole comparative genomic hybridization

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.     

D-mannitol production by resting state whole cell biotrans-formation of D-fructose by heterologous mannitol and formate dehydrogenase gene expression in Bacillus megaterium

An in vivo system was developed for the biotransformation of D-fructose into D-mannitol by the expression of the gene mdh encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides ATCC12291 in Bacillus megaterium. The NADH reduction equivalents necessary for MDH activity were regenerated via the oxidation of formate to carbon dioxide by coexpression of the gene fdh encoding Mycobacterium vaccae N10 formate dehydrogenase (FDH). High-level protein production of MDH in B. megaterium required the adaptation of the corresponding ribosome binding site. The fdh gene was adapted to B. megaterium codon usage via complete chemical gene synthesis. Recombinant B. megaterium produced up to 10.60 g/L D-mannitol at the shaking flask scale. Whole cell biotransformation in a fed-batch bioreactor increased D-mannitol concentration to 22.00 g/L at a specific productivity of 0.32 g D-mannitol (gram cell dry weight)(-1) h(-1) and a D-mannitol yield of 0.91 mol/mol. The nicotinamide adenine dinucleotide (NAD(H)) pool of the B. megaterium producing D-mannitol remained stable during biotransformation. Intra- and extracellular pH adjusted itself to a value of 6.5 and remained constant during the process. Data integration revealed that substrate uptake was the limiting factor of the overall biotransformation. The information obtained identified B. megaterium as a useful production host for D-mannitol using a resting cell biotransformation approach.     

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

Background  

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis.     

Survival of a plasmid-bearing strain of Bacillus subtilis introduced into compost

Survival of Bacillus subtilis strain 168 containing plasmid pAB224, which carries a gene for tetracycline resistance, was studied in mushroom compost under mesophilic and thermophilic conditions. Stable populations of B. subtilis were maintained as spores in both sterile and fresh mushroom compost incubated at 37 degrees C. At 65 degrees C, the introduced B. subtilis populations declined during incubation but spores were still detectable after 28 d. Survival at the higher temperature was greater in fresh than in sterile compost. There was no apparent loss of plasmid pAB224 or plasmid-determined phenotype from the introduced B. subtilis population at either incubation temperature. The frequency of tetracycline resistance in the indigenous Bacillus population was very low (10(-5), but some tetracycline-resistant isolates contained plasmid DNA. Four plasmid DNA profiles were found associated with five Bacillus phenotypes, and some evidence for homology with pAB224 was found. However, pAB224 was found to be a suitable marker for release studies because it was easily recovered, readily distinguished from indigenous plasmids on agarose gels, and was maintained in compost-grown B. subtilis 168 in the absence of any selective pressure.     

High resolution deletion breakpoint mapping in the DMD gene by whole cosmid hybridization.

The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.     

Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization

Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1–D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p?>?0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p?>?0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.     

Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization

Summary We report a general procedure which allows the application of whole cosmid cloned genomic sequences for non-radioactive in situ hybridization. The presence of highly repetitive sequences, like Alu and Kpn fragments, is eliminated through competition hybridization with Cot-1 DNA. The method has been tested and optimized with several randomly chosen cosmids of the human thyroglobulin (Tg) gene (8q24). At present, the procedure can be performed with three of the four tested individual cosmids. In cases where a single clone does not result in a specific signal, a larger fragment may be required, which can be accomplished by using two (partially overlapping) cosmids of the same region. The advantages and further potentialities of such a hybridization approach are discussed.     

Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis

The characteristic shape of bacterial cells is mainly determined by the cell wall, the synthesis of which is orchestrated by penicillin-binding proteins (PBPs). Rod-shaped bacteria have two distinct modes of cell wall synthesis, involved in cell elongation and cell division, which are believed to employ different sets of PBPs. A long-held question has been how these different modes of growth are co-ordinated in space and time. We have now identified the cell division protein, EzrA, and a newly discovered protein, GpsB, as key players in the elongation-division cycle of Bacillus subtilis. Mutations in these genes have a synthetic phenotype with defects in both cell division and cell elongation. They also have an unusual bulging phenotype apparently due to a failure in properly completing cell pole maturation. We show that these phenotypes are tightly associated with disturbed localization of the major transglycosylase/transpeptidase of the cell, PBP1. EzrA and GpsB have partially differentiated roles in the localization cycle of PBP1, with EzrA mainly promoting the recruitment of PBP1 to division sites, and GpsB facilitating its removal from the cell pole, after the completion of pole maturation.     

Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK1

Thermophilic strain JK1 was isolated from compost using xylan as a single carbon source. On the basis of 16S rRNA gene phylogenetic analysis and spo0A gene sequence similarity analysis, strain JK1 was identified as Geobacillus thermodenitrificans strain. During the exponential culture growth, the strain JK1 was found to produce the single xylan degrading enzyme ??45 kDa in size. Xylose was not an inducer of this xylanase. Cloning, expression and characterization of the recombinant xylanase were performed. Xylanase of G. thermodenitrificans JK1 was cellulase-free; pH and temperature optimums were found to be 6.0 and 70°C, respectively. The metal ions Na⁺, K⁺, Ca²⁺, and Co²⁺ showed partial inhibition of the activity, while Mn²⁺ had slight stimulating effect on the enzymatic activity. Recombinant xylanase was thermostable over the temperature range of 55?C70°C. It presented the highest stability after incubation at 55°C for 60 min showing 84% residual activity. 50% residual activity was revealed after incubation at 60°C for 60 min as well as at 65 and 70°C for 30 min. Results of the thermostability experiments showed xylanase of JK1 having quite low thermostability when compared with the respective enzymes of the other geobacilli.