Variations of 18S rDNA Loci Among Six Populations of Paeonia obovata Maxim. （Paeoniaceae） Revealed by Fluorescence In Situ Hybridization

The localization of 18S ribosomal RNA genes （rDNA） by fluorescence in situ hybridization （FISH） had been performed for some species of Paeonla. However, the pattern of 18S rDNA loci among populations Is Indistinct. In the present study, we localized 18S rDNA loci on meiotic or mitotic chromosomes of six populations of Paeonla obovata Maxim. （Paeonlaceae）. Different numbers of rDNA loci were found with different diploid （2n=10） populations, namely eight （Lushl and Mt. JIuhua populations）, 10 （Mt. Talbal population）, and seven （Mt. Guandl population）, whereas tetraplold （2n=20） populations were all found with 16 loci. Aii rDNA loci were mapped near teiomeres of mitotic chromosomes and there was no chromosome with two loci. The present results show that molecular cytological polymorphlsm exists among P. obovata diploid populations, Indicating that structural variations occurred frequently during the evolutionary history of this species, accompanied with differentiation among populations.     

In Situ Cultivation of Subsurface Microorganisms in a Deep Mafic Sill: Implications for SLiMEs

An in situ culturing device was incubated within a flowing borehole in a mafic sill at 1.474 km depth in Evander Au mine, South Africa. The device was designed to enrich methanogenic, Fe^{3 +}-reducing and SO₄ ^{2 ?}-reducing microorganisms using acetate, formate, methanol, Fe^{3 +}-citrate and SO₄ ^{2 ?} enriched agar and sand cartridges. At the end of the 33 day incubation geochemical analyses detected elevated H₂, acetate, CH₄ and Fe concentrations and depleted SO₄ ^{2 ?} concentrations. 16S rDNA sequences and PLFA analyses revealed that the microbial community composition of the substrate-bearing cartridges were distinct from that of the original borehole water and the non-substrate-bearing control cartridge. 16S rDNA and dissimilatory sulfite reductase, dsrAB, gene sequences indicated the device successfully targeted SO₄ ^{2 ?} reducing bacteria (SRB), which were not detected in the original borehole water. 16S rDNA sequences also revealed a shift in the microbial community from one relying on H₂ based methanogenesis to one suggestive of H₂ based acetogenesis supporting aceticlastic methanogenesis and SO₄ ^{2 ?} reduction compatible with the subsurface lithoautotrophic hypothesis.     

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.     

Archaeal Community Revealed by 16s rRNA and Fluorescence in situ Hybridization in a Sulphuric Hydrothermal Hot Spring, Northern Taiwan

Summary The archaeal community composition of Yangmingshan National Park in northern Taiwan was investigated by 16S rRNA and fluorescence in situ hybridization (FISH). Optimization of tetrameric restriction enzyme (TRE) was performed to achieve efficient digestion and differentiation in the restriction fragment length polymorphism (RFLP) fragments, and AciI, BstUI and RsaI were shown to be the optimal TREs for TRE-RFLP. Nine clones were obtained in the studies, with clones M70 and M6 being found to be phylogenetically affiliated to Sulfolobus and Caldisphaera in domain Crenarchaeota, respectively, whereas seven other clones were found to be affiliated to an uncultured and unidentified archaeon isolated from thermoacidic environments. In FISH, soil and water region cells were hybridized with DAPI (4′, 6-diamidino-2-phenylindole) and specific fluorescently labelled probes. 15.69 and 7.16% of the DAPI-stained cells hybridized with universal archaeal probe ARC915 and sulphate-reducing bacterial probe SRB385, respectively.     

通过原位反转录研究MADS盒基因M79的时空表达模式

通过设计可以引发特定mRNA进行转录的特异引物,采用了一种新的原位反转录的方法检测mRNA,这种原位反转录的方法是把原位杂交和反转录进行有机结合的结果,其实用性能和关键步骤都已进行优化。研究表明,只要设立合适的对照原位反转录方法, 检测mRNA的灵敏度高、假阴性和假阳性率低。用这种方法对一个属于AGL2亚组的水稻(Oryza sativa L.)的MADS-盒基因M79的表达进行了分析,结果表明,在水稻从营养到生殖的各个发育阶段,M79均有表达,与平行进行的传统原位杂交的方法得到的结果类似。相对而言,原位反转录法耗时短、容易操作,可以成为一种可靠的原位定位mRNA的基本技术。     

通过原位反转录研究MADS盒基因M79的时空表达模式

通过设计可以引发特定mRNA进行转录的特异引物,采用了一种新的原位反转录的方法检测mRNA,这种原位反转录的方法是把原位杂交和反转录进行有机结合的结果,其实用性能和关键步骤都已进行优化.研究表明,只要设立合适的对照原位反转录方法,检测mRNA的灵敏度高、假阴性和假阳性率低.用这种方法对一个属于AGL2亚组的水稻(Oryza sativa L.)的MADS-盒基因M79的表达进行了分析,结果表明,在水稻从营养到生殖的各个发育阶段,M79均有表达,与平行进行的传统原位杂交的方法得到的结果类似.相对而言,原位反转录法耗时短、容易操作,可以成为一种可靠的原位定位mRNA的基本技术.     

Bacterial diversity and community composition in the chemocline of the meromictic alpine Lake Cadagno as revealed by 16S rDNA analysis

Using different techniques of molecular biology we investigated the bacterial diversity of the chemocline of the meromictic Lake Cadagno. Cloning of a total community 16S rDNA PCR product and subsequent screening with a combination of amplified ribosomal DNA restriction analysis and temporal temperature gradient gel electrophoresis (TTGE) analysis revealed that 30 of 47 randomly selected clones were unique. Partial sequencing and comparative analysis indicated a high bacterial diversity dominated by the gamma-Proteobacteria (33.3%). Most of these rDNA clone sequences were not closely related to any 16S rDNA sequence in the database. In a second approach, the TTGE pattern from an environmental sample was compared with the migration of the cloned 16S rDNA fragments. Four clone types were identified on the environmental pattern by excising and sequencing comigrating bands, three of which were well represented in the library: two Chromatiaceae species and one sequence affiliated with the Desulfobulbus assemblage. Using the fluorescent in situ hybridization technique we essentially confirmed the results of the cloning experiments and the TTGE analysis.