Use of engineered ribozymes to catalyze chimeric gene assembly.

We report the use of engineered ribozymes to shuffle exon cassettes in vitro. Specifically, we have designed derivatives of a group II intron that insert into selected sites in the human tissue plasminogen activator (t-PA) mRNA. The insertion reaction links t-PA sequences to the group II intron sequences so that trans-splicing reactions catalyzed by the intron can be employed to shuffle the t-PA sequences. We expect these results to be generalizable, so that similar ribozymes can be designed to target any desired 13 nucleotide sequence. In principle, the reactions we describe here should be able to link any RNA molecule to any other RNA molecule at any selected point.

Prediction of cis/trans isomerization in proteins using PSI-BLAST profiles and secondary structure information

Background The majority of peptide bonds in proteins are found to occur in the trans conformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt the cis form. Proline cis/trans isomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of proline cis/trans isomerization in proteins would have many important applications towards the understanding of protein structure and function. Results In this paper, we propose a new approach to predict the proline cis/trans isomerization in proteins using support vector machine (SVM). The preliminary results indicated that using Radial Basis Function (RBF) kernels could lead to better prediction performance than that of polynomial and linear kernel functions. We used single sequence information of different local window sizes, amino acid compositions of different local sequences, multiple sequence alignment obtained from PSI-BLAST and the secondary structure information predicted by PSIPRED. We explored these different sequence encoding schemes in order to investigate their effects on the prediction performance. The training and testing of this approach was performed on a newly enlarged dataset of 2424 non-homologous proteins determined by X-Ray diffraction method using 5-fold cross-validation. Selecting the window size 11 provided the best performance for determining the proline cis/trans isomerization based on the single amino acid sequence. It was found that using multiple sequence alignments in the form of PSI-BLAST profiles could significantly improve the prediction performance, the prediction accuracy increased from 62.8% with single sequence to 69.8% and Matthews Correlation Coefficient (MCC) improved from 0.26 with single local sequence to 0.40. Furthermore, if coupled with the predicted secondary structure information by PSIPRED, our method yielded a prediction accuracy of 71.5% and MCC of 0.43, 9% and 0.17 higher than the accuracy achieved based on the singe sequence information, respectively. Conclusion A new method has been developed to predict the proline cis/trans isomerization in proteins based on support vector machine, which used the single amino acid sequence with different local window sizes, the amino acid compositions of local sequence flanking centered proline residues, the position-specific scoring matrices (PSSMs) extracted by PSI-BLAST and the predicted secondary structures generated by PSIPRED. The successful application of SVM approach in this study reinforced that SVM is a powerful tool in predicting proline cis/trans isomerization in proteins and biological sequence analysis.

A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae

Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature- sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP 18 gene has been cloned and its polyadenylated transcript identified.

Efeito da infecção pelo vírus da febre amarela no mecanismo de splicing celular

Pós-graduação em Microbiologia - IBILCE

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^

The role of splicing factors and small nuclear RNAs in spliceosomal formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatiotemporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

The Role of Splicing Factors and Small Nuclear RNAS in Spliceosomal Formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

Electronic states of trans-polyacetylene, poly(p-phenylene vinylene) and sp-hybridised carbon species in amorphous hydrogenated carbon probed by resonant Raman scattering

Inclusions of sp-hybridised, trans-polyacetylene [trans-(CH)x] and poly(p-phenylene vinylene) (PPV) chains are revealed using resonant Raman scattering (RRS) investigation of amorphous hydrogenated carbon (a-C:H) films in the near IR – UV range. The RRS spectra of trans-(CH)x core Ag modes and the PPV CC-H phenylene mode are found to transform and disperse as the laser excitation energy ћωL is increased from near IR through visible to UV, whereas sp-bonded inclusions only become evident in UV. This is attributed to ћωL probing of trans-(CH)x chain inhomogeneity and the distribution of chains with varying conjugation length; for PPV to the resonant probing of phelynene ring disorder; and for sp segments, to ћωL probing of a local band gap of end-terminated polyynes. The IR spectra analysis confirmed the presence of sp, trans-(CH)x and PPV inclusions. The obtained RRS results for a-C:H denote differentiation between the core Ag trans-(CH)x modes and the PPV phenylene mode. Furthermore, it was found that at various laser excitation energies the changes in Raman spectra features for trans-(CH)x segments included in an amorphous carbon matrix are the same as in bulk trans-polyacetylene. The latter finding can be used to facilitate identification of trans-(CH)x in the spectra of complex carbonaceous materials.

Searching the self in Seoul : trans-youth and urban social networking in Korea

This paper presents a brief analysis of Seoul trans-youth’s search for identity through urban social networking, arguing that technological, socio-cultural and environmental (urban) contexts frame how mobility and ubiquity are (re)created in Seoul. The paper is empirically based on fieldwork conducted in Seoul, South Korea, from 2007 to 2008 as part of a research project on the mobile play culture of Seoul trans-youth(a term that will be explained in detail in the following section). Shared Visual Ethnography (SVE) was used as the research method which involved sharing of visual ethnographic data that were created by the participants. More specifically, the participants were asked to take photos, which were then shared and discussed with other participants and the researcher on the photo-sharing service Flickr. The research also involved a questionnaire and daily activity diaries, as well as interviews. A total of 44 Korean transyouths – including 23 females and 21 males – participated in interviews and photo-sharing. The paper draws specifically on the qualitative data from individual and/or group interviews, the total duration of which was 2–2.5 hours for each participant.

The structure of the copper(II) complex with 4,5-dichlorophthalic acid: The 1:1 complex 'adduct' salt trans-tetraaqua(2-carboxy-4,5-dichlorobenzoato)(4,5-dichlorobenzene-1,2-dicarboxylic acid)copper(II) 2-carboxy-4,5-dichlorobenzoate

The unusual (1:1) complex ‘adduct’ salt of copper(II) with 4,5-dichlorophthalic acid (H2DCPA), having formula [Cu(H2O)4(C8H3Cl2O4) (C8H4Cl2O4)] . (C8H3Cl2O4) has been synthesized and characterized using single-crystal X-ray diffraction. Crystals are monoclinic, space group P21/c, with Z = 4 in a cell with dimensions a = 20.1376(7), b =12.8408(4) c = 12.1910(4) Å, β = 105.509(4)o. The complex is based on discrete tetragonally distorted octahedral [CuO6] coordination centres with the four water ligands occupying the square planar sites [Cu-O, 1.962(4)-1.987(4) Å] and the monodentate carboxyl-O donors of two DCPA ligand species in the axial sites. The first of these bonds [Cu-O, 2.341(4) Å] is with an oxygen of a HDCPA monoanion, the second with an oxygen of a H2DCPA acid species [Cu-O, 2.418(4) Å]. The un-coordinated ‘adduct’ molecule is a HDCPA counter anion which is strongly hydrogen-bonded to the coordinated H2DCPA ligand [O… O, 2.503(6) Å] while a number of peripheral intra- and intermolecular hydrogen-bonding interactions give a two-dimensional network structure.

Three-dimensional hydrogen-bonded structures in the guanidinium salts of the monocyclic dicarboxylic acids rac-trans-cyclohexane-1,2-dicarboxylic acid (2:1, anhydrous), isophthalic acid (1:1, monohydrate) and terephthalic acid (2:1, trihydrate).

The structures of bis(guanidinium)rac-trans-cyclohexane-1,2-dicarboxylate, 2(CH6N3+) C8H10O4- (I), guanidinium 3-carboxybenzoate monohydrate CH6N3+ C8H5O4- . H2O (II) and bis(guanidinium) benzene-1,4-dicarboxylate trihydrate, 2(CH6N3+) C8H4O4^2- . 3H2O (III) have been determined and the hydrogen bonding in each examined. All three compounds form three-dimensional hydrogen-bonded framework structures. In anhydrous (I), both guanidinium cations give classic cyclic R2/2(8) N--H...O,O'(carboxyl) and asymmetric cyclic R1/2(6) hydrogen-bonding interactions while one cation gives an unusual enlarged cyclic interaction with O acceptors of separate ortho-related carboxyl groups [graph set R2/2(11)]. Cations and anions also associate across inversion centres giving cyclic R2/4(8) motifs. In the 1:1 guanidinium salt (II), the cation gives two separate cyclic R1/2(6) interactions, one with a carboxyl O-acceptor, the other with the water molecule of solvation. The structure is unusual in that both carboxyl groups give short inter-anion O...H...O contacts, one across a crystallographic inversion centre [2.483(2)\%A], the other about a two-fold axis of rotation [2.462(2)\%A] with a half-occupancy hydrogen delocalized on the symmetry element in each. The water molecule links the cation--anion ribbon structures into a three-dimensional framework. In (III), the repeating molecular unit comprises a benzene-1,4-dicarboxylate dianion which lies across a crystallographic inversion centre, two guanidinium cations and two water molecules of solvation (each set related by two-fold rotational symmetry), and a single water molecule which lies on a two-fold axis. Each guanidinium cation gives three types of cyclic interactions with the dianions: one R^1^~2~(6), the others R2/3(8) and R3/3(10) (both of these involving the water molecules), giving a three-dimensional structure through bridges down the b cell direction. The water molecule at the general site also forms an unusual cyclic R2/2(4) homodimeric association across an inversion centre [O--H...O, 2.875(2)\%A]. The work described here provides further examples of the common cyclic guanidinium cation...carboxylate anion hydrogen-bonding associations as well as featuring other less common cyclic motifs.

Trans digital storytelling : everyday activism, mutable identity and the problem of visibility

Mainstream representations of trans people typically run the gamut from victim to mentally ill and are almost always articulated by non-trans voices. The era of user-generated digital content and participatory culture has heralded unprecedented opportunities for trans people who wish to speak their own stories in public spaces. Digital Storytelling, as an easy accessible autobiographic audio-visual form, offers scope to play with multi-dimensional and ambiguous representations of identity that contest mainstream assumptions of what it is to be ‘male’ or ‘female’. Also, unlike mainstream media forms, online and viral distribution of Digital Stories offer potential to reach a wide range of audiences, which is appealing to activist oriented storytellers who wish to confront social prejudices. However, with these newfound possibilities come concerns regarding visibility and privacy, especially for storytellers who are all too aware of the risks of being ‘out’ as trans. This paper explores these issues from the perspective of three trans storytellers, with reference to the Digital Stories they have created and shared online and on DVD. These examplars are contextualised with some popular and scholarly perspectives on trans representation, in particular embodied and performed identity. It is contended that trans Digital Stories, while appearing in some ways to be quite conventional, actually challenge common notions of gender identity in ways that are both radical and transformative.