Haplotype-specific <Emphasis Type="Italic">MAPT</Emphasis> exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

Background

Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium.

Methods

In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay.

Results

Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion.

Conclusions

We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium.

     

Microbial diversity in acid mineral bioleaching systems of dongxiang copper mine and Yinshan lead–zinc mine

To understand the composition and structure of microbial communities in acid mineral bioleaching systems, the molecular diversity of 16S rDNA genes was examined using a PCR-based cloning approach. A total of 31 Operational Taxonomic Units (OTUs) were obtained from the four samples taken from four different bioleaching sites in Yinshan lead–zinc mine and Dongxiang copper mine in Jiangxi Province, China. The percentages of overlapping OTUs between sites ranged from 22.2 to 50.0%. Phylogenetic analysis revealed that the bacteria present at the four bioleaching sites fell into six divisions, α-Proteobacteria (1.1%), β-Proteobacteria (2.3%), γ-Proteobacteria (30.8%), Firmicutes (15.4%), Actinobacteria (0.3%) and Nitrospira (50.1%). Organisms of genera Leptospirillum, Acidithiobacillus, and Sulfobacillus, which were in Nitrospira, γ-Proteobacteria, and Firmicutes divisions, respectively, were the most dominant. The results of principal component analysis based on the six phylogenetic divisions and biogeochemical data indicated that the microbial community structure of a site was directly related to the biogeochemical characteristic of that site. It follows therefore that sites with similar biogeochemical characteristics were comprised of similar microbial community structures. The results in our study also suggest that the elements copper and arsenic appear to be the key factors affecting the compositions and structures of microbial community in the four bioleaching sites. Zhiguo He, Shengmu Xiao, Xuehui Xie are equally contributed to this work.     

Development of CMV-and TMV-resistant chili pepper: field perfermance and biosafety assessment

Both cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) coat protein (CP) genes have been transferred to chilli pepper (Capsicum annuum var. Longunt) cultivar 8212 by a modified procedure of Agrobacterium tumefaciens-mediated transformation using hypocotyl as the explant. PCR analysis revealed the presence of both CMV and TMV CP genes in at least 11 primary transformants out of 49 kanamycin-resistant chili pepper plants. Ten T1 lines from five independent transformation events were identified as putative homozygous transgenic lines based on the rooting assay of their T2 seedlings on the kanamycin-containing media. Integration and expression of CMV CP and TMV CP transgenes in one of the homozygous line, 16-13, were confirmed bySouthern blot, RT-PCR and western blot analyses. Line 16-13 was highly resistant to infection of homologous CMV and TMV strains in greenhouse conditions when successively challenged with CMV and TMV or challenged with TMV alone.Futhermore, field trials on T2, T3 and T4 progenies of Line 16-13 were performed on scales of 123, 300 and 10,000 plants, respectively, in consecutive years 1996, 1997 and 1998 with the permission of the Chinese government authority. The transgenic plants displayed delayed symptom development and significantly milder disease severity in field conditions when compared to untransformed chili pepper plants, resulting in 47 and 110% increase in pepper fruit yield in surveys conducted in 1997 and 1998 trials, respectively. Finally, quality analysis and biosafety assesment were performed on transgenic chili pepper fruit concurrently with the control fruit, and demonstrated that the transgenic chili pepper fruit is substantially equivalent to the non-transgenic pepper in terms of the quality and biosafety when consumed as a food additive.     

Understanding the properties of aerobic sludge granules as hydrogels

Aerobic sludge granules are larger, denser microbial aggregates than activated sludge flocs with a smoother and more regular surface, which facilitates greater wastewater treatment intensity. Factors important in their growth are still poorly understood, which is an impediment to the construction and operation of full-scale aerobic sludge granule processes. Data in this article obtained with granules treating an abattoir wastewater provide evidence that aerobic sludge granules are hydrogels. The results also demonstrate a method for characterizing macromolecular associations. The rheological profile of these granules was found to be analogous with that of typical polymer gels. Water uptake or swelling reflects an equilibrium between granule elastic modulus and osmotic pressure, whereby uptake is increased by reducing solute concentration or the elastic modulus. A weakening of the extracellular polymeric substance (EPS) matrix as demonstrated with mechanical spectroscopy was induced by several environmental factors including temperature, pH and ionic strength. Uniform and elastic deformation was observed at low strain. Enzymatic degradation studies indicate that proteins and alpha-polysaccharides were the major granule structural materials. The aerobic sludge granules in the current study were therefore protein-polysaccharide composite physical hydrogels. While aerobic sludge granules treating an abattoir wastewater are used as a case study, many of the fundamental principles detailed here are relevant to other granulation processes. The paradigm established in this study can potentially be applied to better understand the formation of aerobic sludge granules and thus overcome a hurdle in the acceptance of aerobic sludge granulation as an alternative to more traditional wastewater treatment processes.     

Case studies of ends‐out gene targeting in Drosophila

Ends‐in and ends‐out gene replacement approaches have been successfully used to disrupt Drosophila genes involved in a variety of biological processes. These methods combine double‐strand breaks and homologous recombination to replace a targeted chromosome region with a designed DNA sequence. Unfortunately, these methods require large numbers of single animal crosses, making them both time consuming and labor intensive. Here, we designed a single complete targeting vector for use in a mass crossing ends‐out gene targeting study. Importantly, our gene targeting method included a balancer chromosome to block endogenous homologous chromosome pairing and to promote pairing between the foreign targeting DNA fragment and the targeted chromosome. This technique provided successful and efficient gene replacement, greatly facilitating the gene knockout procedure. genesis 47:305–308, 2009. © 2009 Wiley‐Liss, Inc.     

Systems biomedicine: It’s your turn —Recent progress in systems biomedicine

The concept of “systems biology” is raised by Hood in 1999. It means studying all components with a systematic view. Systems biomedicine is the application of systems biology in medicine. It studies all components in a whole system and aims to reveal the patho-physiologic mechanisms of disease. In recent years, with the development of both theory and technology, systems biomedicine has become feasible and popular. In this review, we will talk about applications of some methods of omics in systems biomedicine, including genomics, metabolomics (proteomics, lipidomics, glycomics), and epigenomics. We will particularly talk about microbiomics and omics for common diseases, two fields which are developed rapidly recently. We also give some bioinformatics related methods and databases which are used in the field of systems biomedicine. At last, some examples that illustrate the whole biological system will be given, and development for systems biomedicine in China and the prospect for systems biomedicine will be talked about.“     

Intraclass diversification of immunoglobulin heavy chain genes in the African lungfish

Lungfish (Dipnoi) are the closest living relatives to tetrapods, and they represent the transition from water to land during vertebrate evolution. Lungfish are armed with immunoglobulins (Igs), one of the hallmarks of the adaptive immune system of jawed vertebrates, but only three Ig forms have been characterized in Dipnoi to date. We report here a new diversity of Ig molecules in two African lungfish species (Protopterus dolloi and Protopterus annectens). The African lungfish Igs consist of three IgMs, two IgWs, three IgNs, and an IgQ, where both IgN and IgQ originated evidently from the IgW lineage. Our data also suggest that the IgH genes in the lungfish are organized in a transiting form from clusters (IgH loci in cartilaginous fish) to a translocon configuration (IgH locus in tetrapods). We propose that the intraclass diversification of the two primordial gnathostome Ig classes (IgM and IgW) as well as acquisition of new isotypes (IgN and IgQ) has allowed lungfish to acquire a complex and functionally diverse Ig repertoire to fight a variety of microorganisms. Furthermore, our results support the idea that “tetrapod-specific” Ig classes did not evolve until the vertebrate adaptation to land was completed ~360 million years ago.