Cloning and characterization of cytochrome c oxidase subunit I (COXI) in Gobiocypris rarus

In study of gene expression profile in cloned embryos which derived from D. rerio embryonic nuclei and G. rarus enucleated eggs, cytochrome c oxidase subunit I (COXI) of G. rarus, exhibiting difference at expression level between cloned embryos and zebrafish embryo, was cloned. Its full cDNA length is 1654 bp and contains a 1551 bp open reading frame, encoding a 5.64 kDa protein of 516 amino acids. The alignment result shows that mitochondrion tRNA(ser) is co-transcripted with COXI, which just was the 3'-UTR of COXI. Molecular phylogenic analysis based on COXI indicates G. rarus should belong to Gobioninae, which was not in agreement with previous study according to morphological taxonomy. Comparison of DNA with cDNA shows that RNA editing phenomenon does not occur in the COXI of G. rarus.

氨酰-tRNA合成酶结构域的进化研究

氨酰-tRNA合成酶(Aminoacyl-tRNA synthetases, aaRS)是一类在蛋白质生物合成中具有重要作用的酶，它可以活化氨基酸，并与相应的tRNA相识别，使得基因序列能够被精确的翻译成蛋白质序列，保证了生命体的严谨性和多样性。通常，每一类aaRS都包含有一个催化核心结构域(Catalytic central domain, CCD)和一个结合反密码子的结构域(Anticodon-binding domain, ABD)。大量研究显示，细菌与真核生物中的许多aaRS在一些细菌与真核生物中的基因进化机制与模式、氨酰化途径、结构与功能的进化模式等方面往往有着明显的差异。通过对这些差异的深入研究，对于理解蛋白质的结构、功能的进化将是非常有帮助的。虽然，造成这些差异的本质，目前仍不清楚，但是，所有的这些差异似乎提示，在细菌与真核生物的一些基本生命活动过程中的某些方面，可能还存在着目前尚未被人们所认识到的较大差异。 甘氨酰-tRNA合成酶(Glycyl-tRNA synthetase，GlyRS)在基因组中存在着两种寡聚体形式，即α2β2四聚体和α2二聚体。本研究的结果显示，四聚体和二聚体GlyRS的ABD并不同源，而它们的CCD却具有共同的起源。在进化过程中，由于基因的融合，二聚体GlyRS的ABD融合到α亚基上CCD后的C-末端，而四聚体GlyRS的ABD则加在了β亚基的C-末端。通常，同一物种中只存在一种寡聚体形式的GlyRS，但是在Magnetospirillum magnetotacticum基因组中同时存在GlyRS的两种寡聚体形式，并有多个同源的结构域，而这些同源的结构域很可能来源于不同的基因组。二聚体GlyRS存在于细菌、古细菌和真核生物中，而四聚体GlyRS仅在大多数细菌中发现。在从细菌到真核生物的进化过程中，GlyRS可能经历了一个复杂的进化历程。频繁的基因丢失和获得事件导致了GlyRS分布的差异。水平基因转移是四聚体GlyRS进化的一个主要因素。大量的细菌基因水平转移导致四聚体GlyRS基因可在植物中表达，而在动物中形成假基因。 通常，由于aaRS-I和aaRS-II具有不同的结构和催化机制，它们被认为在进化上没有联系。虽然，苯丙氨酰-tRNA合成酶(phenylalanyl-tRNA synthetase, PheRS)属于aaRS-II，但它的催化机制却类似于aaRS-I。结构域的进化分析表明，细菌、古细菌和真核生物的PheRS具有明显不同的结构，因而导致从细菌到真核生物的进化过程中，PheRS和 tRNAPhe间的识别机制发生了变化。序列分析表明，PheRS的结构域(包括CCD、ABD及其它结构域)与aaRS-I的某些结构域同源，因此，在进化上，PheRS是aaRS-II与aaRS-I之间联系的纽带。这些结果表明，在进化的过程中，aaRS-I和aaRS-II可能是由同一个共同的祖先CCD经过可变剪接和插入演化而来的，结构域间的不同组合导致aaRS-I和aaRS-II在结构和催化机制上的显著差异。

Probing lanthanide ions binding on tRNA(Phe) by H-1 NMR

The structure of phenylalanine transfer ribonucleic acid (tRNA(Phe)) in solution was explored by H-1 NMR spectroscopy to evaluate the effect of lanthanide ion on the structural and conformational change. It was found that La3+ ions possess specific effects on the imino proton region of the H-1 NMR spectra for yeast tRNA(Phe). The dependence of the imino proton spectra of yeast tRNA(Phe) as a function of La3+ concentration was examined, and the results suggest that the tertiary base pair G(15). C-48, which is located in the terminal in the augmented dihydrouridine helix (D-helix), was markedly affected by La3+ (shifted to downfield by as much as 0.35). Base pair U-8. A(14) in yeast tRNA(Phe), which are stacked on G(15). C-48, was also affected by added La3+ when 1 similar to 2 Mg2+ were also present. Another imino proton that may be affected by La3+ in yeast tRNA(Phe) is that of the tertiary base pair G(19). C-56. The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances beween 12.6 and 12.2. This base pair helps to anchor the D-loop to the T Psi C loop. The binding of La3+ caused conformational change of tRNA, which is responsible for shifts to upfield or downfield in H-1 NMR spectra.

Effect of lanthanum ions on tRNA(Phe) structure: Imino proton NMR spectroscopy

The effect of lanthanum ions on the structural and conformational change of yeast tRNA(Phe) was studied by H-1 NMR. The results suggest that the tertiary base pair (G-15)(C-48), which was located in the terminal in the augmented dihydrouridine helix (D-helix), was markedly affected by adding La3+ and shifted 0.33 downfield. Based pair (U-8)(A-14), which is associated with a tertiary interaction, links the base of the acceptor stem to the D-stem and anchors the elbow of the L structure, shifted 0.20 upfield. Another imino proton that may be affected by La3+ in tRNA(Phe) is the tertiary base pair (G-19)(C-56). The assignment of this resonance is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.2. This base pair helps to anchor the D-loop to the T psi C loop.

镧离子对tRNA~(Phe)结构的影响:亚胺质子NMR谱

采用1HNMR方法研究了镧离子对酵母tRNAPhe分子结构和构象变化的影响 .结果表明位于扩大二氢尿嘧啶螺旋 (D 螺旋 )的端梢三级碱基对 (G_15) (C_4 8)明显受加入La3+ 的影响 ,向低场位移 0 33;与三级相互作用相关 ,连接D 茎和接受茎起固定L结构转折的 (U_8) (A_14)碱基对向高场位移 0 2 0 ;另一可能受La3+影响的亚胺质子碱基对为 (G_19) (C_56 ) ,由于该碱基对位于高度叠加的 12 6和 12 2之间 ,其归属仅供参考 ,该碱基对有助于D 环对TΨC环的连接.

铕离子对tRNA~(Phe)结构影响的NMR研究

采用ＮＭＲ波谱方法研究了溶液中铕离子对酵母苯丙氨酸转移核糖核酸（ｐｈｅｎｙｌａｌａ－ｎｉｎｅｔｒａｎｓｆｅｒｒｉｂｏｎｕｃｌｅｉｃａｃｉｄ，简称ｔＲＮＡＰｈｅ）结构和构象变化的影响．Ｅｕ３＋离子对ｔＲＮＡＰｈｅ亚胺质子范围的１ＨＮＭＲ谱具有特殊的影响，酵母ｔＲＮＡＰｈｅ亚胺质子谱作为Ｅｕ３＋浓度函数的研究表明位于扩大二氢尿嘧啶螺旋（Ｄ－螺旋）的端梢三级碱基对Ｇ１５·Ｃ４８明显地受加入Ｅｕ３＋的影响（向低场位移０．８５）；堆积在Ｇ１５·Ｃ４８上的Ｕ８·Ａ１４碱基对在存有１～２个Ｍｇ２＋离子下亦受加入Ｅｕ３＋的影响．酵母ｔＲＮＡＰｈｅ中可能受到Ｅｕ３＋影响的另一亚胺质子为Ｇ１９·Ｃ５６三级碱基对，由于Ｇ１９·Ｃ５６的亚胺质子共振位于高度叠加的１２．６与１２．２之间，其归属仅供参考．该碱基对有助于Ｄ－环对ＴΨＣ环的联接．配位Ｅｕ３＋引起ｔＲＮＡ分子构象的变化并且导致一些谱峰向高场或低场位移.

The complete mitochondrial genome of the ridgetail white prawn Exopalaemon carinicauda Holthuis, 1950 (Crustacean: Decapoda: Palaemonidae) revealed a novel rearrangement of tRNA genes

The complete mitochondrial (mt) DNA sequence was determined for a ridgetail white prawn, Exopalaemon carinicauda Holthuis, 1950 (Crustacea: Decopoda: Palaemonidae). The mt genome is 15,730 bp in length, encoding a standard set of 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes, which is typical for metazoans. The majority-strand consists of 33.6% A, 23.0% C, 13.4% G, and 30.0% T bases (AT skew = 0.057: GC skew = -0.264). A total of 1045 bp of non-coding nucleotides were observed in 16 intergenic regions,,including a major A+ T rich (79.7%) noncoding region (886 bp). A novel translocation of tRNA(Pro) and tRNA(Thr) was found when comparing this genome with the pancrustacean ground pattern indicating that gene order is not conserved among caridean mitochondria. Furthermore, the rate of Ka/Ks in 13 protein-coding genes between three caridean species is Much less than 1, which indicates a strong Purifying selection within this group. To investigate the phylogenetic relationship within Malacostraca, phylogenetic trees based oil Currently available malacostracan complete mitochondrial sequences were built with the maximum likelihood and Bayesian models. All analyses based oil nucleotide and amino acid data strongly support the monophyly of Decapoda. The Penaeidae, Reptantia, Caridea, and Meiura clades were also recovered as monophyletic groups with Strong Statistical Support. However, the phylogenetic relationships within Pleocyemata are unstable, as represented by the inclusion or exclusion of Caridea. (C) 2009 Elsevier B.V. All rights reserved.

Camera Canvas: Image Editing Software for People with Disabilities

Poster is based on the following paper: C. Kwan and M. Betke. Camera Canvas: Image editing software for people with disabilities. In Proceedings of the 14th International Conference on Human Computer Interaction (HCI International 2011), Orlando, Florida, July 2011.

Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a CF cell line. Firstly, the most common mutation, ΔF508, was corrected using a pair of existing ZFNs, which cleave in intron 9, and the donor repair plasmid pITR-donor-XC, which contains the correct CTT sequence and two unique restriction sites. HDR was initially determined to be <1% but further analysis by next generation sequencing (NGS) revealed HDR occurred at a level of 2%. This relatively low level of repair was determined to be a consequence of distance from the cut site to the mutation and so rather than designing a new pair of ZFNs, the position of the existing intron 9 ZFNs was exploited and attempts made to correct >80% of CF-causing mutations. The ZFN cut site was used as the site for HDR of a mini-gene construct comprising exons 10-24 from CFTR cDNA (with appropriate splice acceptor and poly A sites) to allow production of full length corrected CFTR mRNA. Finally, the ability to cleave closer to the mutation and mediate repair of CFTR using the latest gene editing tool CRISPR/Cas9 was explored. Two CRISPR gRNAs were tested; CRISPR ex10 was shown to cleave at an efficiency of 15% and CRISPR in9 cleaved at 3%. Both CRISPR gRNAs mediated HDR with appropriate donor plasmids at a rate of ~1% as determined by NGS. This is the first evidence of CRISPR induced HDR in CF cell lines.

Identification of RNA editing in the human exome and development of DARNED DatabaseSF

RNA editing is a biological phenomena that alters nascent RNA transcripts by insertion, deletion and/or substitution of one or a few nucleotides. It is ubiquitous in all kingdoms of life and in viruses. The predominant editing event in organisms with a developed central nervous system is Adenosine to Inosine deamination. Inosine is recognized as Guanosine by the translational machinery and reverse-transcriptase. In primates, RNA editing occurs frequently in transcripts from repetitive regions of the genome. In humans, more than 500,000 editing instances have been identified, by applying computational pipelines on available ESTs and high-throughput sequencing data, and by using chemical methods. However, the functions of only a small number of cases have been studied thoroughly. RNA editing instances have been found to have roles in peptide variants synthesis by non-synonymous codon substitutions, transcript variants by alterations in splicing sites and gene silencing by miRNAs sequence modifications. We established the Database of RNA EDiting (DARNED) to accommo-date the reference genomic coordinates of substitution editing in human, mouse and fly transcripts from published literatures, with additional information on edited genomic coordinates collected from various databases e.g. UCSC, NCBI. DARNED contains mostly Adenosine to Inosine editing and allows searches based on genomic region, gene ID, and user provided sequence. The Database is accessible at http://darned.ucc.ie RNA editing instances in coding region are likely to result in recoding in protein synthesis. This encouraged me to focus my research on the occurrences of RNA editing specific CDS and non-Alu exonic regions. By applying various filters on discrepancies between available ESTs and their corresponding reference genomic sequences, putative RNA editing candidates were identified. High-throughput sequencing was used to validate these candidates. All predicted coordinates appeared to be either SNPs or unedited.

An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.

Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus.

The isoleucyl-tRNA synthetase (ileS) gene was sequenced in toto from 9 and in part from 31 Staphylococcus aureus strains with various degrees of susceptibility to mupirocin. All strains for which the mupirocin MIC was greater than 8 µg/ml contained point mutations affecting the Rossman fold via Val-to-Phe changes at either residue 588 (V588F) or residue 631 (V631F). The importance of the V588F mutation was confirmed by an allele-specific PCR survey of 32 additional strains. Additional mutations of uncertain significance were found in residues clustered on the surface of the IleS protein.