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.

Biochemical characterization and DNA binding properties of the MocR family member GabR, a PLP-dependent transcription factor

GabR è un fattore di trascrizione chimerico appartenente alla famiglia dei MocR/GabR, costituito da un dominio N-terminale elica-giro-elica di legame al DNA e un dominio effettore e/o di oligomerizzazione al C-terminale. I due domini sono connessi da un linker flessibile di 29 aminoacidi. Il dominio C-terminale è strutturalmente omologo agli enzimi aminotransferasici fold-type I, i quali, utilizzando il piridossal-5’-fosfato (PLP) come cofattore, sono direttamente coinvolti nel metabolismo degli aminoacidi. L’interazione contemporanea di PLP e acido γ-aminobutirrico (GABA) a GabR fa sì che questa promuova la trascrizione di due geni, gabT e gabD, implicati nel metabolismo del GABA. GabR cristallizza come un omodimero con una configurazione testa-coda. Il legame con la regione promotrice gabTD avviene attraverso il riconoscimento specifico di due sequenze dirette e ripetute (ATACCA), separate da uno spacer di 34 bp. In questo studio sono state indagate le proprietà biochimiche, strutturali e di legame al DNA della proteina GabR di Bacillus subtilis. L’analisi spettroscopica dimostra che GabR interagisce con il PLP formando l’aldimina interna, mentre in presenza di GABA si ottiene l’aldimina esterna. L’interazione fra il promotore gabTD e le forme holo e apo di GabR è stata monitorata mediante Microscopia a Forza atomica (AFM). In queste due condizioni di legame è stata stimata una Kd di circa 40 ηM. La presenza di GABA invece, determinava un incremento di circa due volte della Kd, variazioni strutturali nei complessi GabR-DNA e una riduzione del compattamento del DNA alla proteina, indipendentemente dalla sequenza del promotore in esame. Al fine di valutare il ruolo delle caratteristiche topologiche del promotore, sono state inserite cinque e dieci bp all’interno della regione spacer che separa le due sequenze ripetute dirette riconosciute da GabR. I significativi cambiamenti topologici riscontrati nel frammento aggiunto di cinque bp si riflettono anche sulla forte riduzione dell’affinità di legame verso la proteina. Al contrario, l’inserzione di 10 bp provoca solamente l’allontanamento delle sequenze ripetute dirette. L’assenza quindi di cambiamenti significativi nella topologia di questo promotore fa sì che l’affinità di legame per GabR rimanga pressoché inalterata rispetto al promotore non mutato. L’analisi del potenziale elettrostatico superficiale di GabR mostra la presenza di una fascia carica positivamente che si estende lungo un’intera faccia della proteina. Per verificare l’importanza di questa caratteristica di GabR nel meccanismo di interazione al DNA, sono stati preparati ed indagati i mutanti R129Q e K362-366Q, in cui la carica positiva superficiale risultava indebolita. L’affinità di legame dei mutanti di GabR per il DNA era inferiore rispetto alla proteina non mutata, in particolar modo nel mutante K362-366Q. Le evidenze acquisite suggeriscono che la curvatura intrinseca del promotore ed il corretto orientamento delle sequenze sulla doppia elica, più della distanza che le separa, siano critici per sostenere l’interazione con GabR. Oltre a questo, la superficie positiva di GabR è richiesta per accomodare la curvatura del DNA sul corpo della proteina. Alla luce di questo, l’interazione GabR-gabTD è un esempio di come il riconoscimento specifico di sequenze, la topologia del DNA e le caratteristiche strutturali della proteina siano contemporaneamente necessarie per sostenere un’interazione proteina-DNA stabile.

Optical control of the spin state of two Mn atoms in a quantum dot

We report on the optical spectroscopy of the spin of two magnetic atoms (Mn) embedded in an individual quantum dot interacting with a single electron, a single exciton, or a single trion. As a result of their interaction to a common entity, the Mn spins become correlated. The dynamics of this process is probed by time-resolved spectroscopy, which permits us to determine an optical orientation time in the range of a few tens of nanoseconds. In addition, we show that the energy of the collective spin states of the two Mn atoms can be tuned through the optical Stark effect induced by a resonant laser field.

50 years of the ‘twin engine’. Franco-German reconciliation, European integration and reflections for Asia. EU Centre Singapore Background Brief No. 8, March 2013

On 22 January 2013, French President François Hollande and German Chancellor Angela Merkel gathered in Berlin to celebrate the 50th anniversary of the signing of the Élysée Treaty, the document that ended centuries of rivalry and warfare between their two countries. It is all too easy to forget the importance of Franco-German reconciliation. The 1950 Schuman Declaration, which led to the creation of the European Union’s (EU) predecessor, the European Coal and Steel Community (ECSC), sought to render the prospect of war between France and Germany ‘not only unthinkable but materially impossible’. Over 60 years later, when the EU was awarded the Nobel Peace Prize, the Norwegian Nobel Committee noted that indeed, ‘war between Germany and France is unthinkable’. Halfway around the world in Asia, the other theatre of World War II, tensions between China and Japan have arisen, with Taiwan and South Korea also in the fray. Nationalist movements in these countries have grown. This background brief lays out the issues for a timely reappraisal of the applicability, or otherwise, of the European integration and reconciliation processes to East Asia. The brief seeks to outline the contours of the historic act of Franco-German reconciliation, and its consequences ever since. Starting from a brief look at the history of rivalry and war between the two countries, the brief examines the events leading to the signing of the Élysée Treaty in 1963, and the development of Franco-German exchanges that have cemented the relationship. Difficulties between the countries are also raised. A timescale analysis of the opinion of the two publics is considered, as a measure of the success of Franco-German reconciliation.

The works of Sir William Temple, bart. : to which is prefixed, the life and character of the author, considerably enlarged.

--I. Life of the author. An essay on the original and nature of government. Observations upon the united provinces of the Netherlands. Letters containing an account of the most important transactions that passed in Christendom from 1665 to 1672.-II. Sequel of the author's letters, serving to supply the loss of the first part of his memoirs. A survey of the constitutions and interests of the empire, Sweden, Denmark, Spain, Holland, France, and Flanders in 1671. A letter to the Duke of Ormond, written in October 1673. Memoirs, pt. II--III.--III. [Essays] Poetry.--IV. Letters to the king, the Prince of Orange, &c. Index.

History of the reign of Philip the Second;

Bibliographical footnotes.

The two angry women of Abingdon,

Series title also at head of t.-p.

The Countess Cosel; a romance of history of the times of Augustus the Strong,

Mode of access: Internet.

The second part of The vision, : a Pindarick ode: occasioned by Their Majesties happy coronation. /

"Advertisement"--P. [1] at end.

The works of the Right Reverend Father in God, Ezekiel Hopkins...now first collected.

I. Account of former editions of Bishop Hopkins's works. Account of the present edition. Life of Bishop Hopkins. Critical remarks on the author's writings. The Vanity of the world. A practical exposition on the Lord's prayer. A catechistical exposition on the Lord's prayer. Exposition on the Ten commandments.--II. Discourses on the law. Discourses concerning sin. The doctrine of the two covenants. The doctrine of the two sacraments. The nature and necessity of regeneration.--III. The all-sufficiency of Christ to save sinners. The excellency of heavenly treasures. Practical Christianity, in working out our own salvation. The assurance of heaven and salvation, a powerful motive to serve God with fear. On glorifying God in His attributes. The almost-Christian discovered. On the nature, corruption, and renewing of the conscience. The great duty of mortification.--IV. Death disarmed of its sting; from several considerations. Miscellaneous sermons. Index of texts illustrated.

The history of the rebellion and civil wars in England /

The "Life" has a special t.p. dated 1842.

A sermon upon the death of the queen preach'd Dec. 18, 1737.

Mode of access: Internet.

Redgauntlet. A tale of the eighteenth century,

Mode of access: Internet.

Lives of the Hanoverian queens of England.

I. Sophia Dorothea of Celle, wife of George I. Caroline of Ansbach, queen of George II.--II. Charlotte Sophia of Mecklenburg-Strelitz, queen of George III. Amelia Elizabeth Caroline of Brunswick, queen of George IV. Adelaide f Saxe Meiningen, queen of William IV.

Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.