eQTL结合ASE分析显示帕金森病相关基因Lrrk2表达受顺式作用调控

富亮氨酸重复激酶2（Lrrk2）基因与家族性及散发性帕金森病(Parkinson′s disease, PD)密切相关,但其作用机制仍不十分清楚.本文以单核苷酸多态性(single nucleotide polymorphism, SNP)为切入点,对其调控机制进行研究. 以近交系C57BL/6J (B6)和DBA/2J (D2)小鼠杂交1代小鼠为实验动物,借助基因表达数量性状基因座(expression quantitative trait loci, eQTL)分析技术,结合等位基因特异性表达（allele specific expression difference, ASE）技术,验证帕金森病相关基因Lrrk2的顺式调控机制. eQTL分析显示,Lrrk2基因所在的位置有1个具有显著统计学意义（LRS值≥20）的顺式调节基因座.针对Lrrk2基因外显子区域错意突变SNP的ASE分析得出： rs50098646位点在cDNA水平上G∶A两碱基比值偏离1∶1,与gDNA水平中G∶A两碱基比值差异显著（P<0.01）,表达受顺式作用调节. 本研究结果表明,Lrrk2基因的表达受到顺式作用的调控.     

Visualization of single fluorophores in living cells

Methods applicable to visualizing single fluorophores in living cells are described, namely, laser epifluorescence, confocal, near-field, two-photon, and total internal reflection microscopy. It is demonstrated that total internal reflection microscopy is the most appropriate for visualizing single fluorophores near the substrate-medium interface. This method can be used for studying receptors, ion channels, and numerous cytoskeletal and signal molecules located on or near the basal cell membrane. It is demonstrated that stringent criteria are necessary when identifying single molecules, as these objects emit a limited number of photons before irreversible photobleaching and their fluorescence is obscured by autofluorescence or out-of-focus fluorescence. The methods used for studying the lateral mobility of single molecules floating on the cell membrane are also described.     

Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs

Four different tandem EGFPs were constructed to elucidate the nuclear microenvironment by quantifying its diffusional properties in both aqueous solution and the nuclei of living cells. Diffusion of tandem EGFP was dependent on the length of the protein as a rod-like molecule or molecular ruler in solution. On the other hand, we found two kinds of mobility, fast diffusional mobility and much slower diffusional mobility depending on cellular compartments in living cells. Diffusion in the cytoplasm and the nucleoplasm was mainly measured as fast diffusional mobility. In contrast, diffusion in the nucleolus was complex and mainly much slower diffusional mobility, although both the fast and the slow diffusional mobilities were dependent on the protein length. Interestingly, we found that diffusion in the nucleolus was clearly changed by energy depletion, even though the diffusion in the cytoplasm and the nucleoplasm was not changed. Our results suggest that the nucleolar microenvironment is sensitive to energy depletion and very different from the nucleoplasm.     

Structure of a capsular polysaccharide isolated from Salmonella enteritidis

Salmonella enteritidis is a food-borne enteric human pathogen that can form a complex protective extracellular matrix. We describe here a component of this matrix which is distinct from other known salmonella extracellular polysaccharides such as cellulose and colanic acid. We have used glycosyl composition and linkage analysis, as well as 1D and 2D NMR spectroscopy to determine the structure of this polysaccharide. We propose that the primary saccharide in the S. enteritidis capsule has a branched tetrasaccharide repeating unit having the following structure: -->3)-alpha-D-Galp-(1-->2)-[alpha-Tyvp-(1-->3)]-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->. This structure is partially substituted on both tyvelose and galactose with a glucose-containing side chain. It further bears considerable similarity to the O antigen from this organism, a feature found in a number of other capsules from Gram-negative bacteria. In addition, we have detected fatty acids at levels that indicate the presence of a lipid anchor.     

Characterization of a novel Photosystem I-LHCI supercomplex isolated from Chlamydomonas reinhardtii under anaerobic (State II) conditions

A novel supercomplex of Photosystem I (PSI) with light harvesting complex I (LHCI) was isolated from the green alga Chlamydomonas reinhardtii. This novel supercomplex is unique as it is the first stable supercomplex of PSI together with its external antenna. The supercomplex contains 256 chlorophylls per reaction center. The supercomplex was isolated under anaerobic conditions and may represent the State II form of the photosynthetic unit. In contrast to previously reported supercomplexes isolated in State I, which contain only 4 LHC I proteins, this supercomplex contains 10-11 LHC I proteins tightly bound to the PSI core. In contrast to plants, no LHC II is tightly bound to the PSI-LHCI supercomplex in State II. Investigation of the energy transfer from the antenna system to the reaction center core shows that the LHC supercomplexes are tightly coupled to the PSI core, not only structurally but also energetically. The excitation energy transfer kinetics are completely dominated by the fast phase, with a near-complete lack of long-lived fluorescence. This tight coupling is in contrast to all reports of energy transfer in PSI-LHCI supercomplexes (in State I), which have so far been described as weakly coupled supercomplexes with low efficiency for excitation energy transfer. These results indicate that there are large and dynamic changes of the PSI-LHCI supercomplex during the acclimation from aerobic (State I) to anaerobic (State II) conditions in Chlamydomonas.     

Phytochromes from Agrobacterium tumefaciens: difference spectroscopy with extracts of wild type and knockout mutants

Phytochromes are photoreceptors that occur in plants, fungi and bacteria, among others in the phytopathogen Agrobacterium tumefaciens. We constructed single and double knockout mutants of the two A. tumefaciens phytochromes Agp1 and Agp2. In liquid culture, the double mutant revealed a reduced growth rate, whereas the growth rates of the single mutants did not differ significantly from that of the wild type. Using these mutants, we analyzed the spectral properties of native A. tumefaciens phytochromes. A wild-type A. tumefaciens cell contains about 10 molecules of Agp1 and about 19 molecules of Agp2. Dark conversion of native Agp1 and Agp2 proceeds from Pfr to Pr and from Pr to Pfr, respectively, as has already been reported for the recombinant proteins. The spectral properties of recombinant and native Agp2 were significantly different. Mixing experiments with extracts from the double mutant and recombinant Agp2 imply that the spectral properties of Agp2 are modulated by components of the extract.     

Exploring mechanochemical processes in the cell with optical tweezers

Force and torque, stress and strain or work are examples of mechanical and elastic actions which are intimately linked to chemical reactions in the cell. Optical tweezers are a light-based method which allows the real-time manipulation of single molecules and cells to measure their interactions. We describe the technique, briefly reviewing the operating principles and the potential capabilities to the study of biological processes. Additional emphasis is given to the importance of fluctuations in biology and how single-molecule techniques allow access to them. We illustrate the applications by addressing experimental configurations and recent progresses in molecular and cell biology.     

Candidate genes for drought tolerance and improved productivity in rice (Oryza sativa L.)

Candidate genes are sequenced genes of known biological action involved in the development or physiology of a trait. Twenty-one putative candidate genes were designed after an exhaustive search in the public databases along with an elaborate literature survey for candidate gene products and/or regulatory sequences associated with enhanced drought resistance. The downloaded sequences were then used to design primers considering the flanking sequences as well. Polymerase chain reaction (PCR) performed on 10 diverse cultivars that involvedJaponica, Indica and local accessions, revealed 12 polymorphic candidate genes. Seven polymorphic candidate genes were then utilized to genotype 148 individuals of CT9993 × IR62266 doubled haploid (DH) mapping population. The segregation data were tested for deviation from the expected Mendelian ratio (1:1) using a Chi-square test (<1%). Based on this, four candidate genes were assessed to be significant and the remaining three, as non-significant. All the significant candidate genes were biased towards CT9993, the female parent in the DH mapping population. Single-marker analysis strongly associated (<1%) them to different traits under both well-watered and low-moisture stress conditions. Two candidate genes,EXP15 andEXP13, were found to be associated with root number and silicon content in the stem respectively, under both well-watered and low-moisture stress conditions     

Distribution and Biology of the New Zealand Endemic Catshark, Halaelurus dawsoni

Synopsis Halaelurus dawsoni has a restricted geographic range, occurring only in south-eastern New Zealand. It is primarily a demersal inhabitant of the upper continental slope, plateaus, and ridges at 250–800 m depth. Halaelurus dawsoni is a voracious carnivore that feeds on a wide variety of crustaceans and fishes. Maximum recorded length is 418 mm total length, and males and females grow to similar maximum lengths. Length at 50% maturity is about 340–350 mm for males and 330–360 mm for females. The reproductive mode of H. dawsoni is single oviparity, with one leathery egg case being carried per uterus. It appears that most embryonic development occurs after egg cases are deposited on the seabed. The reproductive mode of species of Halaelurus in the subgenus Halaelurus is multiple oviparity, whereas for those in the subgenus Bythaelurus it is single oviparity or aplacental viviparity. It has been suggested that single oviparity is a primitive reproductive mode, and that aplacental viviparity evolved from it via the intermediate stage of multiple oviparity. However, the relationship between reproductive mode and Halaelurus subgenus suggests that aplacental viviparity may have evolved directly from single oviparity in the subgenus Bythaelurus without passing through a multiple oviparous stage.