Active transposition in zebrafish.

We have identified a new family of Tc1-like transposons in the zebrafish, Danio rerio. The sequence of a candidate active transposon, deduced from sample Tzf elements, shows limited resemblance to the previously described Tdr1 elements of zebrafish. Both the Tzf and the Tdr elements are extremely abundant in zebrafish. We describe here a general strategy for detecting transposition events in a complex genome and demonstrate its utility by selectively monitoring hundreds of potentially active Tzf copies in the zebrafish genome against a background of other related elements. We have followed members of a zebrafish pedigree, using this two-dimensional transposon display strategy, to identify the first examples of active transposition of such elements in vertebrates.

Cellular stress inhibits transposition of the yeast retrovirus-like element Ty3 by a ubiquitin-dependent block of virus-like particle formation.

Many stress proteins and their cognates function as molecular chaperones or as components of proteolytic systems. Viral infection can stimulate synthesis of stress proteins and particular associations of viral and stress proteins have been documented. However, demonstrations of functions for stress proteins in viral life cycles are few. We have initiated an investigation of the roles of stress proteins in eukaryotic viral life cycles using as a model the Ty3 retrovirus-like element of Saccharomyces cerevisiae. During stress, Ty3 transposition is inhibited; Ty3 DNA is not synthesized and, although precursor proteins are detected, mature Ty3 proteins and virus-like particles (VLPs) do not accumulate. The same phenotype is observed in the constitutively stressed ssa1 ssa2 mutant, which lacks two cytoplasmic members of the hsp70 family of chaperones. Ty3 VLPs preformed under nonstress conditions are degraded more rapidly if cells are shifted from 30 degrees C to 37 degrees C. These results suggest that Ty3 VLPs are destroyed by cellular stress proteins. Elevated expression of the yeast UBP3 gene, which encodes a protease that removes ubiquitin from proteins, allows mature Ty3 proteins and VLPs to accumulate in the ssa1 ssa2 mutant, suggesting that, at least under stress conditions, ubiquitination plays a role in regulating Ty3 transposition.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

The two single-strand cleavages at each end of Tn10 occur in a specific order during transposition.

During Tn10 transposition, the element is excised from the donor site by double-strand cleavages at the two transposon ends. Double-strand cleavage is a central step in the nonreplicative transposition reaction of many transposons in both prokaryotes and eukaryotes. Evidence is presented to show that the Tn10 double-strand cut is made by an ordered, sequential cleavage of the two strands. The transferred strand is cut first, and then the nontransferred strand is cleaved. The single-strand nicked intermediate is seen to accumulate when Mn2+ is substituted for Mg2+ in the reaction or when certain mutant transposases are used. The fact that the transferred strand is cleaved before the non-transferred strand implies that the order of strand cleavages is not the determining factor that precludes a replicative mechanism of transposition.

Evaluation of online tools as didactic resource in the university education

Recently, many efforts have been made in the academic world to adapt the new degrees to the new European Higher Education Area (EHEA). New technologies have been the most important factor to carry out this adaptation. In particular, the tools 2.0 have been spreading quickly, not just the Web 2.0, but even in all the educational levels. Nevertheless, it is now necessary to evaluate whether all these efforts and all the changes, carried out in order to obtain improved academic performance among students, have provided good results. Therefore, the aim of this paper is focused on studying the impact of the implementation of information and communication technologies (ICTs) in a subject belonging to a Master from the University of Alicante in the academic year (2010-2011). In special, it is an elective course called "Advanced Visual Ergonomics" from the Master of Clinical Optometry and Vision. The methodology used to teach this course differs from the traditional one in many respects. For example, one of the resources used for the development of this course is a blog developed specifically to coordinate a series of virtual works, whose purpose is that the student goes into specific aspects of the current topic. Next, the student participates in an active role by writing a personal assessment on the blog. However, in the course planning, there is an attendance to lessons, where the teacher presents certain issues in a more traditional way, that is, with a lecture supported with audiovisual materials, such as materials generated in powerpoint. To evaluate the quality of the results achieved with this methodology, in this work the personal assessment of the students, who have completed this course during this academic year, are collected. In particular, we want to know their opinion about the used resources, as well as the followed methodology. The tool used to collect this information was a questionnaire. This questionnaire evaluates different aspects of the course: a general opinion, quality of the received information, satisfaction about the followed methodology and the student´s critical awareness. The design of this questionnaire is very important to get conclusive information about the methodology followed in the course. The questionnaire has to have an adequate number of questions; whether it has many questions, it might be boring for the student who would pay no enough attention. The questions should be well-written, with a clear structure and message, to avoid confusion and an ambiguity. The questions should be objectives, without any suggestion for a desired answer. In addition, the questionnaire should be interesting to encourage the student´ s interest. In conclusion, this questionnaire developed for this subject provided good information to evaluate whether the methodology was a useful tool to teach "Advanced Visual Ergonomics". Furthermore, the student´s opinion collected by this questionnaire might be very helpful to improve this didactic resource.

The great didactic of John Amos Comenius /

Mode of access: Internet.

English grammar, by lectures: comprehending the principles and rules of syntactical parsing on a new and highly approved system; intended as a text book for students; containing exercises in syntax, rules for parsing by transposition, critical notes, and a lecture on rhetoric. /

Checklist Amer. imprints

The pastoral care. : A didactic poem, in three parts. Addressed to the junior clergy.

Page [6] at front a blank.

... Transposition (keyboard and orchestral)

Mode of access: Internet.

The Gospel of St. John : in Greek and English, interlined, and literally translated; with a transposition of the words into their due order of construction; and a dictionary, defining and parsing them: principally designed for the use of schools /

Mode of access: Internet.

The art of reading : containing a number of useful rules exemplified by a variety of selected and original pieces, narrative, didactic, argumentative, poetical, descriptive, pathetic, humourous, and entertaining, together with dialogues, speeches, orations, addresses, and harangues : calculated to improve the scholar in reading and speaking with propriety and elegance, and to impress the minds of youth with sentiments of virtue and religion : designed for the use of schools and families /

Mode of access: Internet.

A synopsis of didactic theology.

Page 308 incorrectly numbered 803.

Infancy : or, The management of children; a didactic poem in 3 books /

Mode of access: Internet.